Induced Neuronal Cells: How to Make and Define a Neuron

Cell Stem Cell

Review

Induced Neuronal Cells:How to Make and Define a Neuron

Nan Yang,1 Yi Han Ng,1 Zhiping P. Pang,2,4 Thomas C. Sudhof,2,3 and Marius Wernig1,*1Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology2Department of Molecular and Cellular Physiology3Howard Hughes Medical InstituteStanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA4Present address: Child Health Institute of New Jersey and Department of Neuroscience and Cell Biology, Robert Wood Johnson MedicalSchool, 89 French Street, New Brunswick, NJ 08901, USA*Correspondence: [email protected] 10.1016/j.stem.2011.11.015

Cellular plasticity is a major focus of investigation in developmental biology. The recent discovery thatinduced neuronal (iN) cells can be generated from mouse and human fibroblasts by expression of definedtranscription factors suggested that cell fate plasticity is much wider than previously anticipated. In thisreview, we summarize the most recent developments in this nascent field and suggest criteria to help defineand categorize iN cells that take into account the complexity of neuronal identity.

IntroductionSomatic cell nuclear transfer and in vitro induction of pluripo-

tency in somatic cells by defined factors provided unambiguous

evidence that the epigenetic state of terminally differentiated

somatic cells is not static and can be reversed to amore primitive

one (Gurdon, 2006; Jaenisch and Young, 2008; Yamanaka and

Blau, 2010). Inspired by these results, stem cell biologists have

recently identified approaches to directly convert fibroblasts

into induced neuronal (iN) cells, indicating that direct lineage

conversions are possible between very distantly related cell

types (reviewed in Vierbuchen and Wernig, 2011). Importantly,

iN cells can also be derived from defined endodermal cells.

The reprogramming process both induces neuronal properties

and extinguishes prior donor cell identity, and therefore repre-

sents a complete and functional lineage switch as opposed to

generation of a chimeric phenotype. Since the discovery in the

late 1980s that MyoD, a key regulatory transcription factor in

the skeletal muscle lineage, can induce many features of muscle

cells in fibroblasts, several other examples of remarkable cell-

fate changes have been observed in response to forced expres-

sion of transcriptional regulators, but until recently it was

assumed that this phenomenon is limited to closely related cell

lineages (for discussion and historical background, see Graf,

2011, in this issue of Cell Stem Cell). After the initial description

of iN cells, additional studies showed that fibroblasts can be

directly converted to a diverse range of cell types, such as cardi-

omyocytes (Ieda et al., 2010), blood cell progenitors (Szabo

et al., 2010), and hepatocytes (Huang et al., 2011; Sekiya and

Suzuki, 2011). In this review, we discuss the recent advances

in direct lineage reprogramming toward the neuronal lineage

and propose criteria that can be used to identify successfully

reprogrammed iN cells.

Direct Conversion of Mouse Fibroblasts to NeuronsAs transcription factors play key determining roles in cell fate

specification, we hypothesized that forced expression of

a combination of such factors may be sufficient to directly

convert mouse fibroblasts into neuronal cells (Vierbuchen

et al., 2010). Following lentiviral expression of 19 candidate

genes, we detected cells with neuronal morphologies and

expression of neuronal markers, which suggests that such

a conversion may indeed be possible. A systematic evaluation

of different combinations revealed that the five transcription

factors Ascl1, Brn2, Olig2, Zic1, and Myt1l are the most critical

genes for this process. Out of those five factors, the pool of

Brn2, Ascl1, and Myt1l (BAM) was shown to be sufficient to

induce neuronal cells that exhibited both molecular and all prin-

cipal functional properties of neurons (Figure 1). Surprisingly, the

conversion efficiency of embryonic fibroblasts was estimated to

be close to 20% within 2 weeks, indicating that the conversion

toward neuronal fates is substantially faster and more efficient

than iPSC formation. Also in contrast to induction of pluripo-

tency, iN cell reprogramming does not require cell proliferation,

which is arguably a more favorable condition for epigenetic

changes and a key mediator of iPSC reprogramming (Hong

et al., 2009; Kawamura et al., 2009; Li et al., 2009; Marion

et al., 2009; Utikal et al., 2009).

This first iN cell study raised many new questions that the field

has now begun to address. Among other things, it was unclear

what the exact cell of origin for iN cells was and whether endo-

dermal cells could be coaxed toward an iN cell fate. From

a developmental perspective, it remained unresolved whether

the reprogramming involved an intermediate neural progenitor

cell state, how similar iN cells are compared to bona fide

neurons, whether iN cells possessed a regional identity, and if

modifying the combination of transcription factors would bestow

a specific neural subtype identity. Chromatin biologists would be

interested to know how completely the epigenetic landscape is

remodeled toward a neuronal pattern and whether the reprog-

ramming factors initiate the neurogenic programwhile suppress-

ing the original cell fates or whether iN cells retain molecular

‘‘memories’’ of their cell of origin. In relation to potential transla-

tional application, questions also remained about whether iN

cells can functionally integrate into the brain, and last but not

Cell Stem Cell 9, December 2, 2011 ª2011 Elsevier Inc. 517

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http://dx.doi.org/10.1016/j.stem.2011.11.015

Figure 1. Summary of Induced Neural Lineage Cells to DateSeveral proof-of-principle studies have established the possibility of generating different subtypes of induced neuronal cells as well as neural precursor cells frommouse and/or human fibroblasts. Diagrammed here are examples from mouse and human fibroblasts to excitatory (although iN cells expressing inhibitory-neuron-specific markers can be occasionally detected by multiple groups, or IPSCs can even be recorded from iN cells cocultured with mouse glial cells [Yooet al., 2011], the main majority of iN cells produced are excitatory cells), dopaminergic, andmotor iN cells. Marro et al. (2011) also described the direct conversionfrommouse hepatocytes to excitatory iN cells. The parentheses indicate alternative reprogramming factors from different studies that result in the same neuronalsubtype.

Cell Stem Cell

Review

least, whether methods could be developed to generate human

iN cells. In the past few months a number of reports have

provided the first answers to some of these questions.

A Direct Endoderm-to-Ectoderm SwitchIn order to address some of the outstanding questions, we

looked at converting definitive endodermal cells into iN cells

(Marro et al., 2011). Intriguingly, we found that the exact same

three reprogramming factors were sufficient to induce iN cells

from primary liver cultures as from fibroblasts. Taking advantage

of a well-characterized Albumin-Cre allele, we unequivocally

confirmed that Albumin-expressing hepatocytes were the origin

of iN cells, thereby demonstrating that transcription-factor-

mediated lineage reprogramming is possible across major

lineage boundaries. We were also able to assess the transcrip-

tional network dynamics during reprogramming and to compare

the expression profile of fibroblast- and hepatocyte-derived iN

cells. These results indicated that the timing of the two reprog-

ramming processes is different and that hepatocytes appear to

be more resistant to the lineage switch. Moreover, we also

explored how thoroughly iN cells are reprogrammed. We found

that the donor-cell-type-specific expression signatures were

robustly silenced in both fibroblast- and hepatocyte-derived iN

cells. Thus it seems that the exact same three transcription

factors not only can induce a neuronal program, but can also

downregulate two unrelated donor transcriptional programs.

Detailed gene expression analysis at a population- and single-

cell-level indicated that iN cells possess a limited degree of

epigenetic memory of their donor cells, but these transcriptional

remnants decreased over time. It will be interesting to investigate

the molecular mechanism underlying the transcriptional

518 Cell Stem Cell 9, December 2, 2011 ª2011 Elsevier Inc.

silencing that occurs, and to see whether it is similar to mecha-

nisms that are used during cell fate specification in the embryo.

Given the substantial differences between the fibroblast and liver

transcriptional programs, it seems unlikely that the transcrip-

tional silencing is directly mediated by the neuronal transcription

factors themselves. Nevertheless, it is a formal possibility that

the BAM factors target and inhibit a large number of key

lineage-determining factors representing many nonneuronal

cell fates. Alternatively, the mutual lineage switch could be

caused by a more general mechanism. Perhaps when cells are

becoming specified to one particular lineage a process becomes

activated that leads to transcriptional silencing of many other

lineage programs. For example, lineage-determining factors

may have to compete for a finite amount of certain ubiquitously

expressed and required cofactors, which would lead to suppres-

sion of undesired lineages once differentiating cells have

committed to one lineage. E-proteins could potentially be one

such critical cofactor as they are known to heterodimerize with

many different lineage-specific bHLH transcription factors (Mas-

sari and Murre, 2000).

iN Cells from Human FibroblastsAnother important question that remained open after our initial

publication was whether iN cells could also be generated from

human fibroblasts. This issue is important because potential clin-

ical applications could only be realized with human cells. As the

exact same four transcription factors can reprogram bothmouse

and human fibroblasts into iPSCs, one might have expected that

converting human fibroblasts to iN cells could be achieved in the

same way as that used for mouse fibroblasts. However, when

the BAM factors were introduced into human fetal fibroblasts,

Cell Stem Cell

Review

the resulting cells remained immature and failed to generate

action potentials when depolarized (Pang et al., 2011). This

finding was later confirmed by another group that reported little

reprogramming by the BAM factors in human cells, and in this

case, attributed to pronounced cell death (Qiang et al., 2011).

We therefore screened 20 additional factors in combination

with BAM and found that by introducing the bHLH factor Neu-

roD1, the generation of functional neuronal cells from human

fibroblasts can be achieved. These human iN cells expressed

a variety of neuronal markers including Tuj1, MAP2, NeuN, Neu-

rofilament, and Synapsin and exhibited functional neuronal

properties as judged by the measurement of action potentials.

Moreover, when cultured with primary cortical neurons, both

spontaneous and evoked postsynaptic currents could be de-

tected in these cells, demonstrating their synaptic maturation.

However, the first functional synapses were found only after 5–

6 weeks, suggesting that the full maturation of human iN cells

is a slow process. The same four factors could also convert post-

natal foreskin fibroblasts into synaptically competent iN cells

with comparable timing and efficiency. Like mouse iN cells,

most of the human iN cells expressed mRNAs characteristic of

glutamatergic neurons, such as vGLUT1 and vGLUT2. After

downregulation of the exogenous transcription factors, the iN

cells retained their stability, which indicated that an intrinsic

program was established to maintain the newly adopted

neuronal identity. The overall efficiency of generating human iN

cells with four factors (2%–4%) was about 10-fold lower than

that of mouse with just three factors (compare to Vierbuchen

et al., 2010). The observed species differences in the iN cell re-

programming may appear unexpected in light of the robustness

of generating human iPSCs. However, upon closer inspection,

the two different human reprogramming paradigms do share

many similarities. The drop in human iPSC reprogramming effi-

ciency as compared to mouse is of a similar magnitude, espe-

cially when taking into account that only a small fraction of plated

fibroblasts and only a small subset of these cells’ progenies are

forming iPSCs. Similarly, it takes much longer for iPSC-like colo-

nies to appear with human fibroblasts as compared to those of

mouse. Thus, human cells in general appear to be less plastic

and have a higher epigenetic ‘‘hurdle’’ for reprogramming to

both iN cells and iPSCs. Finally, human ESC-derived neurons

require amounts of time to develop synaptic competence that

are similar to those of human iN cells (Johnson et al., 2007; Wu

et al., 2007). Thus, a long maturation time may be an inherent

property of human cells, which is perhaps not surprising given

that human brain development is orders of magnitude slower

than that of rodents.

In attempts to convert adult human fibroblasts to neurons,

another group turned to using the five factors that we initially

found in mouse to be the most critical of the 19 tested candidate

factors. The resulting cells possessed a series of neuronal prop-

erties including certain functional properties such as the ability to

generate action potentials when depolarized (Qiang et al., 2011).

The acquisition of more mature functional properties such as

synaptic transmission was less clear. This observation is similar

to ours when adult fibroblasts were infected with BAM and

NeuroD1 (Pang et al., 2011). Nevertheless, iN cells were gener-

ated from Familial Alzheimer’s Disease (FAD) patients with muta-

tions in PSEN genes and were found to exhibit disease-specific

traits, providing important proof-of-concept that iN cells can be

used to model human disease. Specifically, the FAD-iN cells

showed the presence of amyloid precursor protein (APP) puncta

in endosomes, which was not readily detected in the originating

FAD fibroblasts. This phenotype could be rescued by overex-

pression of wild-type PSEN1. Of note, one of the reprogramming

factors used in this study is Olig2, another member of bHLH

family. Olig2 is not specific to neurons and can promote both

neuronal and oligodendroglial fates depending on the develop-

mental context (Mizuguchi et al., 2001; Novitch et al., 2001;

Zhou et al., 2001; Lu et al., 2002; Park et al., 2002; Takebayashi

et al., 2002). However, in contrast to Ascl1 and NeuroD1, Olig2 is

thought to act as a repressor, and to associate with Ngn2 and

E47 to antagonize their neurogenic effect (Lee et al., 2005).

Future work will need to elucidate the function of these transcrip-

tion factors during reprogramming. As a number of different tran-

scription factor combinations can induce neuronal cells, there

may be several parallel pathways to the neuronal lineage. Alter-

natively, the different transcription factors may eventually acti-

vate the same core program to induce neuronal identity.

The fact that the vast majority of reprogramming factors

known to date are transcriptional regulators is not surprising

given their ability to efficiently activate gene expression, and it

also fits with the idea that this gene class contains ‘‘master regu-

lators’’ and ‘‘terminal selectors’’ for specific lineages (Weintraub

et al., 1989; Hobert, 2011). It is surprising, however, thatmiRNAs,

which are thought to function predominantly through downregu-

lation of gene activity, seem to be very powerful agents to

mediate reprogramming. Two independent groups have recently

derived human and mouse iPSCs by adding miRNAs in the

absence of any additional transcription factors (Anokye-Danso

et al., 2011; Miyoshi et al., 2011). Similarly, Yoo et al. (2011)

showed that by introducing miR-9/9* and miR-124, human fibro-

blasts can be reprogrammed into cells with neuron-likemorphol-

ogies expressing the panneuronal marker MAP2. While these

phenotypic changes are truly remarkable, the miRNAs alone

were not sufficient to induce functional iN cells. However, the

addition of the transcription factors NEUROD2, ASCL1, and

MYT1L greatly increased the conversion efficiencies and led to

the formation of iN cells from fetal and adult human fibroblasts

with all the major functional properties of neurons, including

synapse formation. Intriguingly, this report also underscored

the essential role of bHLH transcription factors for generation

of human iN cells. miR-9* and miR-124 are specifically ex-

pressed in postmitotic neurons and were shown to repress the

expression of SWI/SNF complex subunit Baf53a. When neural

progenitor cells exit the cell cycle and differentiate into neurons,

Baf53a is replaced by Baf53b and this switch is functionally rele-

vant (Yoo et al., 2009). Therefore, one possibility was that the

miRNAs facilitated reprogramming through promoting this BAF

complex subunit switch. However, prolonging the expression

of BAF53a did not abolish the conversion from fibroblasts to

neurons, and therefore downregulation of this miRNA target

does not seem to be critical in this context (Yoo et al., 2011).

As we showed iN cell induction by NeuroD1, Ascl1, Myt1l, and

Brn2, it appears that the miRNAs are able to replace the tran-

scription factor Brn2 (Pang et al., 2011). However, this idea

was not tested directly, however, and it is possible that the

miRNAs work through yet another mechanism. More recently,


Cell Stem Cell

Review

another group also found miR-124 to be beneficial for human iN

cell formation (Ambasudhan et al., 2011). In this report the

miRNAwas combinedwith BRN2 andMYT1L, further supporting

the idea the miRNAs have a complementary function to Brn2.

Surprisingly, no bHLH transcription factors were used in the

latter report, but the cells also appeared less completely reprog-

rammed based on the absence of convincing evidence for

synaptic competence. Future studies on the miRNA-transcrip-

tion factor interplay responsible for iN cell formation could also

be relevant to regular neural development. Thus, this extremely

nonphysiological method of reprogramming could perhaps

even become a discovery tool for understanding normal devel-

opment.

Induction of Specific Subtypes of Mouse and HumaniN CellsFor clinical and experimental use of iN cells, it would be desirable

to develop ways to generate neurons with neurotransmitter- and

region-specific phenotypes. Liver and fibroblast iN cells gener-

ated with the same reprogramming factors displayed properties

characteristic of excitatory neurons. While this property does not

in itself imply the acquisition of a particular region-specific

phenotype, it could mean that the glutamatergic fate is a default

fate as has been suggested for ESC differentiation systems (Tro-

pepe et al., 2001; Gaspard et al., 2008). Alternatively, the choice

of transcription factors could have specifically induced an excit-

atory subtype. Therefore, the question arises of whether inclu-

sion of subtype-specific transcription factors in the reprogram-

ming cocktail could direct cells into other desired subtypes.

This hypothesis was elegantly tested by Son et al., who gener-

ated iN cells with motor neuron identity directly from fibroblasts

(Son et al., 2011) (Figure 1). Starting out with a fairly large pool of

transcription factors critical for motor neuron specification, they

eventually found that four factors (Lhx3, Hb9, Isl1, and Ngn2) in

combination with the BAM factors generated Hb9-positive

neurons with an efficiency of up to 10% from MEFs. Gene

expression analyses indicated that these induced motor

neuronal (iMN) cells resemble the embryonic and ESC-derived

motor neurons in transcription profiles. Besides displaying elec-

trophysiological properties akin to those of motor neurons, these

iMN cells also formed functional synaptic connections with my-

otubes. When transplanted to the developing chick spinal cord,

most of the iMN cells were engrafted in the ventral horn of the

spine with axons projecting into the ventral roots. In addition,

the cells behaved similarly to ESC-derived motor neurons in

disease conditions. When cultured with glia carrying the G93A

mutation in the Superoxide dismutase (Sod1) gene, a mutation

found in familial forms of amyotrophic lateral sclerosis (ALS),

the survival of iMN cells decreased. Vice versa, iMN cells derived

from Sod1G93A MEFs also showed reduced survival when

cultured with wild-type glia. These first translational studies

suggest that iMN cells can be used as a tool to understand the

pathophysiology of ALS. As a first step in this direction, Son

et al. also infected human ESC-derived fibroblast-like cells

with the seven transcription factors in combination with Neu-

roD1. This approach yielded neuronal cells that could fire action

potentials and expressed Hb9 and vesicular ChAT. More work is

needed to investigate whether iMN cells can be generated from

primary human fibroblasts.


Another clinically relevant neuronal subtype that has been

under intense investigation is the group of midbrain dopami-

nergic (DA) neurons, which are preferentially affected in Parkin-

son’s disease. Recently, two important proof-of-principle

studies described the generation of iN cells expressing tyrosine

hydroxylase (TH), the rate-limiting enzyme in catecholamine

biosynthesis (Caiazzo et al., 2011; Pfisterer et al., 2011). Pfisterer

et al. showed that Lmx1a and Foxa2, when used in conjunction

with the BAM pool, are capable of generating iN cells expressing

TH; Aromatic L-amino acid decarboxylase (AADC), another

crucial enzyme in catecholamine biosynthesis; and importantly,

Nurr1, a marker of midbrain identity. However, the cells did not

express other midbrain markers and were not able to release

dopamine into the media. Another report by Caiazzo et al.

demonstrated the generation of mouse iN cells with dopami-

nergic features by expression of the transcription factors

Ascl1, Nurr1, and Lmx1a. The fraction of TH-positive cells was

reported to be 18% based on a TH-EGFP transgenic reporter

line. The TH-positive cells coexpressed vesicular monoamine

transporter 2 (VMAT2), dopamine transporter (DAT), aldehyde

dehydrogenase 1a1 (ALDH1A1), and calbindin. In contrast to

the BAM/Foxa2/Lmx1a cells described by Pfisterer et al., the

Ascl1/Nurr1/Lmx1a iN cells were able to release dopamine as

determined by amperometry and HPLC analysis, indicating

that the cells possessed an important functional property of

dopamine neurons. Intriguingly, similar results could be obtained

using human fibroblasts and the same reprogramming factors.

However, the cells generated in this study did not express any

regional markers specific to midbrain and displayed immature

morphologies. Moreover, the authors did not investigate whether

the cells were competent to receive synaptic input. Therefore,

despite the use of midbrain dopamine neuron-specific transcrip-

tion factors for reprogramming, only generic dopamine neuron

and no midbrain-specific features were observed, suggesting

incomplete reprogramming. Similarly, genome-wide transcrip-

tional profiling showed substantial differences between the re-

programmed and brain-derived dopamine neurons.

As a cautionary note, the absence of midbrain character is

a critical limitation for clinical application, since only ‘‘authentic’’

human midbrain dopamine neurons are able to restore function

in animal models of Parkinson’s disease (Kriks et al., 2011; Roy

et al., 2006; Yang et al., 2008). Therefore, yet another group

very recently attempted to generate iN cells that are more remi-

niscent of midbrain dopamine neurons (Kim et al., 2011b). This

time transcription factor combinations were screened to induce

EGFP fluorescence in Pitx3:EGFP knockin fibroblasts, a locus

highly specific for midbrain dopamine neurons. Surprisingly,

EGFP-positive cells were readily detected with a combination

of two factors, Ascl1 and Pitx3. Complementation with another

four factors (Nurr1, Lmx1a, Foxa2, and En1) as well as the

patterning factors Shh and FGF8 further enhanced the induction

of Pitx3. The EGFP-positive cells also expressed the generic

dopamine neurons markers TH, DAT, AADC, and VMAT2 and

were able to release dopamine. However, when tested in vivo,

the cells only partially restored dopamine function, and when

a series of midbrain markers were analyzed, both the two-factor

and the six-factor iN cells failed to reach similar transcription

levels found in embryonic or adult midbrain dopamine neurons.

This finding leads to the somewhat sobering conclusion that

Figure 2. Direct versus Indirect ReprogrammingLong expression of the four Yamanaka factors leads to iPSC formation whenthe fibroblasts are grown in ESCmedia (route 1); whereas the short expressioninduces fibroblasts to a transient, unstable pluripotent state that can be quicklydifferentiated into neural precursors or cardiomyocytes depending on themedia components (route 4). Direct reprogramming (e.g., iN cell reprogram-ming) does not involve a pluripotent intermediate stage (route 3). Futurestudies may demonstrate the possible direct conversion from fibroblasts toneural precursor cells (route 2). Green arrows: reprogramming; gray arrows:differentiation.

Cell Stem Cell

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even six transcription factors may still not be sufficient to fully

reprogram fibroblasts to this specific neuronal subtype.

Directly generating terminally differentiated neurons could be

useful in disease modeling and transplantation studies.

However, a clear limitation of postmitotic iN cells is their inability

to expand once reprogrammed. Large numbers of cells will be

required for cell-replacement-based therapies in a clinical

setting, or for drug screening. Therefore, it would be desirable

to induce expandable neural precursor cells directly from fibro-

blasts. Recently, Ding and colleagues successfully converted

mouse fibroblasts to induced neural precursor cells (iNPCs)

(Kim et al., 2011a). In this study, the recipe for reprogramming

was not tailored to the target cell type, but was instead identical

to the iPSC reprogramming factors (Figure 2). However, unlike as

in iPSC formation, the factors were induced only for a short time,

and the cells were then exposed to media favoring the growth of

neural progenitor cells. After optimization of timing and culture

conditions, colonies that closely resembled neural rosette cells

appeared and expressed several relevant markers. Upon spon-

taneous differentiation, these iNPCs could give rise to multiple

neuronal subtypes and astrocytic cells, indicating that the iNPCs

were at least bipotential neural precursor cells, but cells with oli-

godendrocytic characteristics were not seen; notably, a very

similar approach was also used to generate cardiomyocytes

and blood progenitor cells from fibroblasts (Szabo et al., 2010;

Efe et al., 2011). In these studies, the authors concluded that

the observed transdifferentiation bypassed an intermediate

pluripotent stage because no Oct4 transcripts were observed

in the cell population and the short reprogramming factor

expression was not compatible with iPSC generation. However,

as the same approach can generate multiple somatic as well as

pluripotent lineages, the simplest explanation is that short-term

expression of the pluripotency reprogramming factors indeed

induces a transient pluripotent state, but this state is unstable

and prone to differentiation, and cannot be stabilized by environ-

mental cues only (Figure 2). Future studies will address whether

a ‘‘direct’’ conversion between fibroblasts and neural progeni-

tors is possible using neural transcription factors (Figure 2).

Although this approach is intriguing, the efficiency of forming

rosette-like colonies appeared low and the cells could not be

expanded well. It remains to be seen what the similarities and

differences of these indirect and direct iNPCs will be and

whether such unstable pluripotent cells can also be generated

from human fibroblasts.

Defining Criteria for iN CellsIn recent months there has been a wave of reports published

describing various methods to generate iN cells of various sorts.

Given the infancy of the field, different criteria were applied to

define converted neuronal cells, complicating the direct compar-

ison of the different approaches. Consistent standards would be

helpful, and we would therefore like to suggest here a panel of

criteria that can be used to define iN cells with various degrees

of reprogramming. First, we propose the term ‘‘induced neuronal

cells (iN cells)’’ as opposed to ‘‘induced neurons,’’ to contrast re-

programmed cells with brain-derived cells. Second, we propose

that the term ‘‘iN cell’’ should only be endorsedwhen the extent of

reprogramming can be documented as being reasonably

complete. In a nutshell, we believe fully reprogrammed iN cells

should have a distinct neuronal morphology, express neuron-

specific gene products, and exhibit the two principal functional

properties of neurons: action potentials and synaptic transmis-

sion. This level of validation would be equivalent to that used for

in-vitro-generated neurons from neural or embryonic stem cells

in previous work (Song et al., 2002; Vicario-Abejon et al., 2000;

Wernig et al., 2004). Cells exhibiting only a subset of these prop-

erties should be termed ‘‘partially reprogrammed iN cells.’’ Since

the reprogramming process mimics neuronal maturation, the

term ‘‘immature iN cells’’ could be used alternatively. We would

like to point out, however, that at a practical level it is difficult to

distinguish these two conceptually very different interpretations.

Despite the great diversity of neurons in the nervous system,

there are a number of typical properties shared by the vast

majority of neurons. First, neurons have common morphological

features. They are characterized by cellular polarization and typi-

cally extend multiple arborizing dendrites and one single axon

from the cell body (soma). Because of these unique structural

properties, neurons also express specific cytoskeletal proteins

such as neurofilaments, microtubules, and microtubule-associ-

ated proteins. Second, neurons have unique membrane charac-

teristics, with the presence of numerous constitutively open,

voltage-gated, or transmitter-dependent ion channels and intra-

cellular second messenger-regulated metabotropic receptors.

Together, these proteins confer the passive and active

membrane properties of neurons, such as the ability to generate

action potentials. Finally, neurons are characterized by their


Figure 3. Neuronal Properties in Order ofStringency (Maturation/Extent of Reprogramming)Diagrammed here are characteristic properties of neuronsand the specific criteria and assays that can be applied toevaluate iN cell maturation. The degree of reprogrammingincreases from top to bottom as indicated by the shadedtriangle.

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ability to form synapses, which are specialized cell-cell contacts

between neurons where neurotransmission takes place. It is

important to note that not all neurons receive synaptic input

from other neurons (e.g., primary sensory neurons), but every

neuron has output function and in most cases forms synapses

with other neurons (one exception are neuromodulatory neurons

such as some dopamine neurons, which are believed to not form

classical synapses).

Building on the generic properties of neurons, we would like to

propose criteria to define a fully reprogrammed iN cell (Figure 3).

Similar to the maturation process of cultured primary neurons,

reprogramming fibroblasts gradually and asynchronously

acquire the full set of neuronal properties over time (Vierbuchen

et al., 2010). Therefore, at any given time there will be a heteroge-

neous population containing iN cells that are reprogrammed to

different degrees and thus display a range of neuronal features.

Figure 3 shows such properties in the order of stringency, which

is also roughly the order of appearance in reprogrammed iN cells

or neurons during development. These properties, therefore, can

serve as criteria to help define the degree of reprogramming or

the maturation stage of iN cells.

The earliest notable changes of converted iN cells aremorpho-

logical, with the generation of a round soma protruding one small

and thin process (Vierbuchen et al., 2010, see also Figure 4B).

Gradually, more processes are generated, which begin to

branch. At first all neurites express both axonal and dendritic

markers, but early during differentiation one process gains the

characteristics of an axon whereas the remaining neurites

become dendrites (e.g., MAP2 versus Tau; see Silverman

et al., 2001). During the early stages of neurogenesis, newly

born neurons become immunoreactive for the nuclear epitope

NeuN (Mullen et al., 1992) and Poly-Sialated Neural Cell Adhe-

sion Molecule (PSA-NCAM) (Bonfanti et al., 1992). Note that

the presence of Tuj1 or MAP2 reactivity in neuritic extensions

of a cell does not necessarily mean that the respective cell is

actually a neuron.

During iN cell reprogramming, both passive and active

membrane properties gradually approach the levels seen in

primary cultured neurons (Vierbuchen et al., 2010). The resting

membrane potential becomes more hyperpolarized and the

capacitance increases as the cell volume increases. The input

resistance also decreases, presumably as a result of the appear-


ance of more neuronal membrane channel

proteins. As for passive membrane properties,

under current-clamp recording mode, action-

potential-like responses induced by depolariza-

tion grow to resemble a mature stereotypic

shape with increasing amplitudes and narrow-

ing widths. A mature, all-or-none action poten-

tial is characterized by a constantly high ampli-

tude irrespective of the induction and current, with a fast

depolarization and fast repolarization. The rapid repolarization

ensures the regeneration of voltage-gated Na+ channels, which

enables mature cells to fire trains of action potentials in rapid

succession (Koester and Siegelbaum, 2000; Lockery et al.,

2009). Therefore, the presence of repetitive action potentials is

a clear sign that the ion channels responsible for generating

action potentials are properly orchestrated. Repetitive action

potentials occur spontaneously or can be evoked by injecting

currents. However, the presence of spontaneous action

potentials does not necessarily mean that the cells are receiving

excitatory synaptic input, as both spontaneous membrane fluc-

tuations and various pace-making channels (e.g., hyperpolariza-

tion-activated cyclic nucleotide-gated channels) can cause

spontaneous firing. The development of passive and active

membrane properties is facilitated by, but not dependent on,

the presence of glial cells (Wu et al., 2007). By contrast, however,

the formation of functional synapses requires factors secreted

by glial cells (Banker, 1980; Eroglu and Barres, 2010; Wu et al,

2007). Morphological evidence of synapse formation can be ob-

tained by high-resolution fluorescence microscopy showing

a presynaptic vesicle protein such as synapsin, synaptophysin,

or synaptotagmin localized in small puncta in close proximity

to MAP2-positive dendrites. Electron microscopic analysis and

demonstration of a postsynaptic density as well as synaptic vesi-

cles in the presynaptic axonal compartment can provide even

stronger evidence. Morphology alone does not prove the exis-

tence of functional synapses, but unambiguous evidence of

synaptic transmission can be obtained using electrophysiolog-

ical tools (Regehr and Stevens, 2001). A prerequisite for synaptic

transmission is the expression of certain ionotropic neurotrans-

mitter receptors, which can be tested by exposing the cells to

specific receptor agonists such as glutamate or GABA and

determining the current responses in the presence and absence

of channel blockers to show specificity. The expression of neuro-

transmitter receptors is still not proof of function. The presence

of functional synapses can be unambiguously demonstrated

by the recording of typical spontaneous postsynaptic currents

(PSCs) with sharp rise and slow decay phases caused by both

the dynamics of presynaptic vesicle exocytosis and the biophys-

ical properties of postsynaptic receptors (Figures 4D and 4E).

PSCs can be recorded spontaneously or evoked by stimulation

Figure 4. Examples of Morphological and Electrophysiological Criteria of iN Cells(A) Tuj1-positive fibroblasts extending one or more thin cellular process.(B) Immature iN cells extending one or two long branching neurites from their soma.(C) More mature iN cell morphologies characterized by multiple, long, branching processes extending from the cell body. Often iN cells sit on top of a densenetwork of neurites derived from surrounding cells.(D) A voltage-clamp recording of spontaneous PSCs. The baseline is fairly tight and there is only little noise detectable. Both clusters of spikes (region 1, black)and separated spikes (region 2, red) representmost likely postsynaptic events as seen by higher time resolution (lower black and red traces). PSCs are typically ofasymmetric shape with a fast deviation from the baseline followed by a slow rectification.(E) A trace in the same recording mode with a much noisier baseline. Region 1 shows a group of spikes with amplitude deviation similar to that in (D). Higherresolution (lower black trace) reveals high levels of baseline fluctuation precluding the identification of PSCs. Other areas in the same trace (e.g., region 2, redtrace) contain spikes that most likely represent synaptic currents.

Cell Stem Cell

Review

of presynaptic terminals (e.g., by extracellular stimulation). A part

from the differences in kinetics between excitatory and inhibitory

PSCs, application of specific neurotransmitter receptor blockers

can demonstrate specificity of the responses and distinguish

inhibitory (IPSC) from excitatory (EPSC) postsynaptic activity.

Sometimes, noise caused by fluctuation of membrane channels

during the recording can cause membrane potential deflections

that appear to be synaptic currents (Figure 4D1). Therefore, not

every deviation from the baseline is mediated by synaptic trans-

mission, and cautious interpretation of the recorded traces is

essential. The evaluation of presynaptic competence is in our

view not only the most rigorous criterium of neuronal function,

but is also the most difficult test, as it requires either the culture

of pure iN cells in a sufficient density or paired recordings.

Finally, mature synapses also often show simple short-term

plasticity such as depression or facilitation.

In summary, we propose distinguishing fully and partially re-

programmed iN cells based on a specific combination of molec-

ular and functional characteristics. We suggest that the critical

feature of fully reprogrammed iN cells should be the demonstra-

tion of synaptic competence as determined by the presence

of spontaneous and evoked presynaptic and postsynaptic

responses in mixed or pure neuronal cultures. iN cells can also

be assessed either in vitro or after transplantation into rodent

brains, which may provide a better environment for maturation.

Outlook and Concluding RemarksAlthough still in a nascent stage, the field of direct somatic

lineage reprogramming has already attracted a lot of attention.

From a biological standpoint, it may become a newmethodology

in the developmental and molecular biology toolbox. It offers

a new way to interrogate transcription factor function indepen-

dent of the physiological environment, and to study the complex

interplay between sequence-specific transcriptional regulators

and various repressive and active chromatin states, as well

as the recruitment of their underlying chromatin-modifying

enzymes. Moreover, the generation of iN cells represents a novel

way to study the mechanisms of cell fate decisions of neural

development and postmitotic neuronal maturation. In addition,

the use of human iN cells provides an avenue for studying human

developmental processes in live cultures, which may enable the

discovery of species-specific differences relative to the much-

better-studied model organisms.

From a medical point of view, direct lineage reprogramming

provides an alternative, potentially complementary tool to

many of the proposed applications of iPSC technology for both

disease modeling and development of cell-based therapies.

Recently, several elegant reports of assessing disease-related

phenotypes in iPSC-derived neurons have provided an impor-

tant proof-of-principle that at least some cellular aspects of

complex brain diseases can be recapitulated with patient-

derived cells in vitro (Marchetto et al., 2010; Brennand et al.,

2011; Nguyen et al., 2011). Both iPSC and iN cell approaches

are complicated by heterogeneity with respect to maturation

and presumably subtype specification. Especially iPSCs have

shown a substantial line-to-line variability with regards to differ-

entiation potential (Hu et al., 2010). Future studies will show

whether directly generated iN cells can provide a better repre-

sentation of the cellular variability, which might in turn simplify

the discovery and analysis of disease-associated phenotypes.


Cell Stem Cell

Review

Moreover, the generation of iN cells from a large cohort of

patients appears quite feasible, whereas the generation and

neuronal differentiation of iPSCs would be a very cumbersome

and slow process.

For potential use in regenerativemedicine, both iPS and iN cell

approaches could provide autologous neuronal donor cells for

transplantation. Expandability is obviously a major advantage

of iPSCs over postmitotic iN cells and may very well be a limiting

factor. However, the postmitotic state of iN cells would have the

advantage of a much lower risk of cancer and teratoma forma-

tion. Integration-free iN cells would be preferable for clinical

use. Along those lines, it will be exciting to see whether small

molecules can be found to replace some or all transcription

factors, similar to the recent successes in iPSCs. Finally, future

studies will need to improve iN cell generation from adult human

fibroblasts since the current low efficiencies represent another

hurdle for any of these translational applications.

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Induced Neuronal Cells: How to Make and Define a Neuron

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